Formation of an instantaneous nick for highly efficient adenylation of oligonucleotides by ligase without subsequent jointing.

By forming a nick at the adenylation site instantaneously, nucleic acids are efficiently adenylated by T4 DNA ligase. The subsequent ligation is successfully suppressed in terms of rapid conversion of the instantaneous nick to a more stable gap. It is helpful to understand enzymatic ligation dynamics, and the adenylated products can be used for various practical applications.

Unexpected Mechanism and Inhibition Effect for Nonspecific Amplification Involving Dynamic Binding of Primers with Background DNA.

Nonspecific amplification is a serious issue in DNA detection as it can lead to false-positive results and reduce specificity. It is very important to well understand its mechanism through sequencing nonspecific products. Here, an approach is developed using a nanopore sequencing technique after acquiring the long repetitive sequence of DNA products from nonspecific amplification. Based on the sequencing results, a new mechanism of nonspecific amplification designated as dynamic mismatched primer binding (DMPB) with the background DNA (bgDNA) is proposed. Unexpectedly, our findings show that the primers (∼20 nt) can bind to bgDNA for primer extension when only 6-11 fully matched (9-14 mismatched) base pairs are formed. After the single-stranded DNAs (ssDNAs) attached to the first primer are produced, more interestingly, with the aid of DNA polymerase, the other primer can bind to these ssDNAs in the case that the fully matched base pairs formed between them are even shorter than 6 bp. As a result, perfect "seeds" for polymerase chain reaction with information on both primers are produced so that exponential nonspecific amplification can occur. The DMPB mechanism can explain nonspecific amplification in other approaches as well. Finally, a mini-hairpin DNA is used to effectively inhibit nonspecific amplification by preventing the formation of an unexpected primer-bgDNA complex.

A novel screening method of DNA methylation biomarkers helps to improve the detection of colorectal cancer and precancerous lesions.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies, and early detection plays a crucial role in enhancing curative outcomes. While colonoscopy is considered the gold standard for CRC diagnosis, noninvasive screening methods of DNA methylation biomarkers can improve the early detection of CRC and precancerous lesions. METHODS: Bioinformatics and machine learning methods were used to evaluate CRC-related genes within the TCGA database. By identifying the overlapped genes, potential biomarkers were selected for further validation. Methylation-specific PCR (MSP) was utilized to identify the associated genes as biomarkers. Subsequently, a real-time PCR assay for detecting the presence of neoplasia or cancer of the colon or rectum was established. This screening approach involved the recruitment of 978 participants from five cohorts. RESULTS: The genes with the highest specificity and sensitivity were Septin9, AXL4, and SDC2. A total of 940 participants were involved in the establishment of the final PCR system and the subsequent performance evaluation test. A multiplex TaqMan real-time PCR system has been illustrated to greatly enhance the ability to detect precancerous lesions and achieved an accuracy of 87.8% (95% CI 82.9-91.5), a sensitivity of 82.7% (95% CI 71.8-90.1), and a specificity of 90.1% (95% CI 84.3-93.9). Moreover, the detection rate of precancerous lesions of this assay reached 55.0% (95% CI 38.7-70.4). CONCLUSION: The combined detection of the methylation status of SEPT9, SDC2, and ALX4 in plasma holds the potential to further enhance the sensitivity of CRC detection.

Enhanced antibacterial behavior of a novel Cu-bearing high-entropy alloy.

Contact infection of bacteria and viruses has been a critical threat to human health. The worldwide outbreak of COVID-19 put forward urgent requirements for the research and development of the self-antibacterial materials, especially the antibacterial alloys. Based on the concept of high-entropy alloys, the present work designed and prepared a novel Co0.4FeCr0.9Cu0.3 antibacterial high-entropy alloy with superior antibacterial properties without intricate or rigorous annealing processes, which outperform the antibacterial stainless steels. The antibacterial tests presented a 99.97% antibacterial rate against Escherichia coli and a 99.96% antibacterial rate against Staphylococcus aureus after 24 h. In contrast, the classic antibacterial copper-bearing stainless steel only performed the 71.50% and 80.84% antibacterial rate, respectively. The results of the reactive oxygen species analysis indicated that the copper ion release and the immediate contact with copper-rich phase had a synergistic effect in enhancing antibacterial properties. Moreover, this alloy exhibited excellent corrosion resistance when compared with the classic antibacterial stainless steels, and the compression test indicated the yield strength of the alloy was 1015 MPa. These findings generate fresh insights into guiding the designs of structure-function-integrated antibacterial alloys.

Prevalence of hereditary breast and ovarian cancer (HBOC) predisposition gene mutations among 882 HBOC high-risk Chinese individuals.

Identification of deleterious variants in hereditary breast and ovarian cancer (HBOC) susceptibility genes allows for increased clinical surveillance and early detection, and could predict the response to poly (ADP-ribose) polymerase (PARP) inhibitor in patients with advanced ovarian carcinomas. To determine the prevalence and clinical prediction factors for HBOC syndrome, 882 selected individuals underwent multigene panel testing for HBOC risk assessment during the period from January 2015 to March 2018. Overall, 176 deleterious mutations were observed in 19.50% (n = 172) of individuals. Twenty-six of 176 mutations could not be retrieved in related public databases and were considered to be novel. Among patients with ovarian cancer, 115 deleterious mutations were identified in 429 patients (48.6%) with significant enrichment for a family history of breast or ovarian cancer syndrome (P < .05). In the breast cancer subgroup, 31 deleterious mutations were identified in 261 patients. Besides BRCA1 (8; 25.8%) and BRCA2 (11; 35.5%), the most frequently occurring genes, an additional 12 deleterious mutations (38.7%) were found in seven other susceptibility genes. Higher mutation incidence (57.9%) was observed in subjects with histories of breast and ovarian cancer. Our results highlighted the genetic heterogeneity of HBOC and the efficiency of a multigene panel in carrying out risk assessment.